S. Aydogan Turkoglu et al. / Hacettepe J. Biol. & Chem., 2018, 46 (3), 329–336

The Bioinformatic and Expression Analysis of PSMD4

PSMD4 Geninin Ekspresyonu ve Biyoinformatik Analizi

Research Article

Sümeyye Aydogan Turkoglu*, Gizem Güler, Feray Köçkar Department of Molecular Biology and Genetics, Faculty of Science and Literature, Balikesir University, Balıkesir, Turkey.

ABSTRACT

6S non-ATPase subunit 4 (PSMD4) that has a molecular weight of 41 kDa is included on 2chromosome 1 (1q21.3). PSMD4 protein is a subunit of the 19S regulatory region in the 26S proteasome. The proteasome breaks down proteins that are not needed or non-functional in the cell. We have limited data about the regulation of this gene in the literature. In our research, changes in the mRNA and protein levels of the PSMD4 gene were investigated in different cell lines (prostate, breast, colon, cervix, hepatoma, osteosarcoma and pancreas) and a normal cell (human vein endothelial cell). The highest expression of PSMD4 gene was seen in HUVEC and PC-3 cells. Bioinformatic analysis was also performed on PSMD4 protein for different species namely human, mouse and rat. Our Bioinformatic analyses showed that first 250 nucleotides are much conserved in all three species.

Key Words PSMD4, cancer, cell lines, bioinformatics.

ÖZ

6S proteazom non ATPaz subunit 4 (PSMD4) kromozom 1’in q kolunun 21.3 bölgesinde lokalize olmuştur ve 41 2Kda moleküler ağırlığında protein kodlamaktadır. PSMD4, 26S proteazomun yapı birimi olan 19S düzenleyici yapı biriminde görev yapmaktadır. Proteozomun ana görevi ihtiyaç duyulmayan işlevini yitirmiş ya da zarar görmüş proteinlerin yıkımıdır. Literatürde çok işlevli protein olan PSMD4 ve onun regülasyonu hakkındaki bilgiler oldukça sınırlıdır. Bu çalışmada; PSMD4’ün mRNA ve protein seviyesindeki ekspresyonu çok sayıda kanser hücre hattında (prostat, meme, kolon, servik, hepotoma, kemik ve pankreas kanseri) ve bir normal hücrede (Göbek kordonu epitel hücresi) incelenmiştir. PSMD4 mRNA ve protein seviyesindeki ekspresyonu PC-3 ve HUVEC hücre hatlarında en yüksek seviyede elde edilmiştir. Ayrıca PSMD4 proteinin insan, fare ve tavşan gibi farklı türlerdeki biyoinformatik analizleri 3 türde ilk 250 amino asit benzer olduğunu ve korunduğu göstermiştir.

Anahtar Kelimeler PSMD4, kanser, hücre hattı, biyoinformatik.

Article History: Received: Aug 21, 2017; Revised: Feb 12, 2018; Accepted: Jul 31, 2018; Available Online: Oct 4, 2018. DOI: 10.15671/HJBC.2018.241 Correspondence to: S. Aydogan Turkoglu, Dept. of Mol. Biol. and Gen., Fact. of Sci. and Literature, Balikesir Uni., Balıkesir, Turkey. Tel: +90 266 612 1278 Fax: +90 266 612 1200 E-Mail: [email protected] 330 S. Aydogan Turkoglu et al. / Hacettepe J. Biol. & Chem., 2018, 46 (3), 329–336

INTRODUCTION mRNA and protein expression of PSMD4 gene fter losing the functional activity of proteins in in various cancer cells, namely, PC-3 and DU-145, Athe cell, proteins are required for demolishing. i.e. Prostate cancer cells; MG-63 and Saos-2, i.e. In eukaryotic cells, these proteins are destroyed Human Osteosarcoma; MCF-7, i.e. Human Breast using lysosomal and proteasomal pathways [1]. Carcinoma; Hela, i.e. Human Cervical cancer; The greatest destruction pathway is HT-29, i.e. Human Colon Carcinoma; PANC, i.e. located in the nucleus and the cytoplasm and Pancreatic cancer; Hep-3B, i.e. Hepatocellular degraded unneeded or damaged proteins. cancer, and in a normal cell, HUVEC, i.e. Human Proteasomes are present in all eukaryotic cells, in Umbilical ENDOTHELIAL. Taken that PSMD4 gene archaea and also in some bacterial cells [2]. encodes three different functional proteins, it is important to analyse the structure of gene and Proteasomes are responsible for destroying protein sequence amongst the species. Therefore, proteins that have completed their function. we also analysed gene and protein sequences of Proteins to be digested should be distinguished human, mouse and rat PSMD4 in a bioinformatical from those that will not be destroyed. To solve manner. this problem, is marked by binding to proteins to be destroyed. The 26S proteasome MATERIALS and METHODS 19S regulatory tag opens the folds by recognizing Materials ubiquitin-labelled proteins and sends them All chemicals used in the studies are suitable proteasome for destruction [1]. The cytosolic 26S for molecular biology studies. Molecular biology proteasome composed of the 20S proteasome and materials, chemicals and used in RT- the 19S is used to degrade the polyubiquitinated PZR studies were obtained from Biochrom, one and is a short-lived and unneeded protein Sigma, Merck, Fermentas, Thermo Scientific in mammals [3]. PSMD4 (26S proteasome non- and Promega. Primers used in the study were ATPase subunit 4) gene encodes an ubiquitin purchased from Macrogen. Detailed information binding protein S5a which serves the 19S about the cell lines used in the study can be found regulatory complex and also exists in the soluble in our previous publications [6-8]. An antibody form in the cytosol. PSMD4 gene also encodes against PSMD4 was bought from Santa Cruz (sc- ASF (Anti-secretory Factor) and Angiocidin. The 53425). β-actin (sc-81178) and anti-mice IgHRP, S5a is a protein located in the proteasomal subunit i.e. A5441 were purchased from Sigma-Aldrich. 26S and linked to the polyubiquitinated chains by Primers used in this research were obtained from two ubiquitin binding motifs. The ASF protein is Macrogen. similar in sequence to S5a but is quite different concerning function. It regulates intestinal fluid Cell Culture that is induced by cholera toxin, and Angiocidin All cells used in the research were grown in is a protein that is over-expressed in various solid DMEM medium (Invitrogen) containing 2 mM tumours and tumour-capillary endothelial cells. L-Glutamine and 10% heat inactivated (56°C for Angiocidin has an anti-angiogenic activity and 1 h) Fetal Calf Serum. Cells were monitored for inhibits tumour growth [4]. Angiocidin protein growth by placing the incubator containing 5%

sequence has a high-level homology with S5a and CO2 at 37°C. Control of cell viability was carried ASF. The carboxy-terminal region of angiocidin out under Trypan Blue Microscopy. The cell lines carries an additional three amino acids, unlike the were grown and routinely passaged twice a week other two proteins, S5a and ASF [5]. [6].

PSMD4 gene is a multifunctional protein, RNA Preparation and Semi Quantitative RT- but there is no cancer-related regulation about PCR this gene in the literature. Therefore, the Cells were grown in 25 cm2 flasks and were purpose of the present research is identifying centrifuged at 10,000 rpm for 5 minutes to expression level of this gene in different cancer obtain pellets. Cell pellets were used for RNA cells and a normal cell model. We analyzed the isolation. Isolation was performed from pellets S. Aydogan Turkoglu et al. / Hacettepe J. Biol. & Chem., 2018, 46 (3), 329–336 331 using the Thermo Scientific RNA isolation kit. Membranes were then blocked in solution (1xTris RNAs were stored at -80°C in the freezer for buffered saline (TBS) containing 5% (w/v) fatless long periods. RT-PCR was used in two separate milk-powder and 0.1% (v/v) Tween-20) to prevent steps. In the first step, reverse transcriptase non-specific binding. Membranes were first (RT) was used for cDNA synthesis. In the second treated with primary antibody PSMD4 (Santa step, amplification of the gene region and Cruz, sc-53425) for 1h at room temperature, and Polymerase Chain Reactions (PCR) were carried after three times of washing, with secondary out by using cDNA obtained from the first step antibody (HRP-conjugated anti-mouse) for 1h with gene specific primers. The PCR conditions (room temperature). Membranes were treated and the primer sequences for PSMD4 are given with chemiluminescence substrate (Pierce) and in Table 1 and 2. PCR products were run on a 1% transferred to film. Correct size bands were (w/v) agarose gel and analyzed in the Gel-Doc analysed with markers. The Image J Software was system. Densitometric analyses were performed used to analyse the bands. The PSMD4 protein with Image J, and the expression of PSMD4 was amount was normalised by the amount of β-Actin normalized by Hb2 (β-2 microglobulin) expression. protein.

Bioinformatic Analysis Western Blot The mRNA and protein sequences of mouse Proteins of each cell lines were extracted by (NM_001282017.1), human (NC_000001.11) and rat applying RIPA buffer. The 50 μg, protein extract, (NM_031331.1) PSMD4 gene were obtained from was applied on a 10% polyacrylamide gel and NCBI database. Mouse, Human and Rat PSMD4 was transferred to PVDF membranes (Millipore). gene sequences were compared by BioEdit v.7.2.5 Table 1. Primer information used for the research. program.

Primer Primer Accession Product Temperature GC (%) Name sequence no size (0C)

PSMD4 5’ GAAGGTGGCAAGATG GTGTTGGAAA3’ NM_002810.2 380bp 48 56 Forward

PSMD4 5’ TCCTTCTCATTGTCCTCCACTGGGCT 3’ NM_002810.2 380bp 53.8 56 Reverse

Hβ-2 5’ TTTCTGGCCTGGAGGCTATC 3’ NM_004048 314bp 55 52 Forward

Hβ-2

5’ CATGTCTCCATCCCACTTAACT 3’ NM_004048 47.6 52 314bp Reverse

Table 2. PCR conditions for PSMD4. . Temperature Steps Time (sec) Cycle (°C)

1 94⁰ 180 1

2 94⁰ 45

56⁰ 45 35

72⁰ 45

3 72⁰ 10 1 332 S. Aydogan Turkoglu et al. / Hacettepe J. Biol. & Chem., 2018, 46 (3), 329–336

RESULTS and DISCUSSION they were tested in our experiments. The PCR products were visualized on an agarose gel, and Expression Analyses of PSMD4 in Selected the bands in the gel were analyzed by Image J Cells program (Figure 1). As seen in Figure 1, the PSMD4 mRNA expression level was analyzed maximum PSMD4 expression was obtained from using Semi-quantitative Reverse Transcription- PC3 and HUVEC cells. Polymerase Chain Reaction (RT-PCR) strategy. The sq-rt-PCR analysis was performed with PSMD4 Protein Analysis in PC-3, HUVEC, specific primers to PSMD4 (Table 1). In expression SAOS-2, HEP3B Cells by Western Blotting We investigated the protein level of PSMD4 gene analysis, the Hβ-2 gene from housekeeping was used to normalize our gene values. In our in the cells with highest PSMD4 mRNA expression laboratory, there are specific primers of this using the Western Blotting Method. Determining gene that we frequently use in our analyzes, and of the protein level of PSMD4 provides relevant

Figure 1: Analysis of mRNA level of PSMD4 gene in different cell lines. Products were obtained with RT-PCR and run on agarose gel. The bands were analysed with Image J. PSMD4 mRNA level was normalized to HB2 level.

Figure 2: The Western Blot Technique was used for analysis at the PSMD4 protein level in PC-3, HUVEC, SAOS-2, HEP3B cancer cell lines. Image j was used to analyse the bands. β-Actin was used for normalisation. S. Aydogan Turkoglu et al. / Hacettepe J. Biol. & Chem., 2018, 46 (3), 329–336 333 information about the comparison of the mRNA proteasome via ubiquitin-like domains and level. PSMD4 protein was analysed in PC-3, ubiquitin-associated domains. In human, there are HUVEC, Saos-2, Hep3B cells by Western Blotting. two different protein variants of PSMD4. PSMD4 The mRNA expression Human prostate cancer variant 1 and angiocidin have 380aa, whereas cells (PC-3) and HUVEC cells have the maximum PSMD4 variant 2 has 377 aa with the exemption protein level of PSMD4 gene. of 255-257 amino acids. The role of these three additional amino acids is not known. They could In silico Analysis of Human, Mouse, and Rat play roles for differential activity of the protein. PSMD4 Protein Our Bioinformatics analyses showed that the first Homo sapiens PSMD4 gene is 1332 bp in length. It 250 nucleotides were much conserved in all three is placed on 1, and contains 10 exons. species (Figure 3). S5a and ASF have identical Mus musculus PSMD4 gene is 1344 bp in length. It 377 amino acid (aa) sequences, while Angiocidin is placed on chromosome 3, and contains 10 exons. is described as having an additional three amino Ratus norvegicus PSMD4 gene is 1334 in length. It acid sequence, namely, Gly255Glu256Arg257 in is placed on chromosome 2. Accession numbers of its C-Terminus. mRNA of these genes are; Homo sapiens PSMD4 gene NC_000001.11, Mus musculus PSMD4 gene DISCUSSIONS NM_001282017.1, Ratus norvegicus PSMD4 gene In the cell, protein must be destructed after losing NM_031331.1. PSMD4 sequence analysis of the its functional activity or when it is damaged. In different species has shown us that there are eukaryotic cells, there are two pathways for some differences between selected mammalian destruction, lysosomal or proteasomal pathways species. For example, human and mouse protein [1]. Proteasome (a multisubunit complex) sequences are 96% similar, whereas human and plays important roles in the regulation of proteins rat protein sequences are 99% identical. There controlling cell-cycle progression and . is also a 97% similarity between mouse and rat For this reason, it has become an important sequences. target for anticancer therapy. Before the degradation of the protein, this specific protein In a human PSMD4 protein, essential for is firstly flagged for destruction by the ubiquitin ubiquitin-binding sites, LALAL and IAYAM conjugation system (which, in the end, results in amino acids are detected in 216-220; 287-291, the attachment of a polyubiquitin chain to the respectively (Figure 3). In human sequence, 197- targeted protein [2]. There are many types of the 262 amino acids are also related to ubiquitin proteasome in a eukaryotic cell. Most specifically interacts with ubiquitylated proteins and in mammals is the cytosolic 26S proteasome

Figure 3: In Silico Analysis of PSMD4 Proteins in Different Species: Essential for ubiquitin-binding, *:197-262aa Ubiquitin is associated with ubiquitin and interacts with ubiquitinated proteins and proteasome via ubiquitin-like domains, Angiocidin has an additional three amino acids, GER). 334 S. Aydogan Turkoglu et al. / Hacettepe J. Biol. & Chem., 2018, 46 (3), 329–336

and it is approximately 2000 kDa. Center of 26S PSMD4 (26S proteasome non-ATPase subunit proteasome has one core 20S particle and two S5a) encodes one of the three proteins that serve regulatory subunits [9]. the 19S regulatory complex. PSMD4 gene also encodes ASF and Angiocidin. The protein S5a Another element in proteasomal degradation is placed in the 26S proteasome subunit. It is a is the amino acid sequences contained in proteins. major protein polyubiquitin recognition domain. Some amino acid sequences make proteins On the other hand, anti-secretory factor, a more vulnerable to degradation and determine protein whose sequence is identical to S5a, is a the half-life of the protein. These are the ‘PEST’​ protein that regulates intestinal fluid transport series. The PEST sequence is a peptide chain rich which is induced by cholera toxin. More important in Proline, Glutamic acid, Serine, and Theronin. than this, Angiocidin is a protein that is expressed This sequence is associated with proteins having excessively in various solid tumours and tumour a short half-life in the cell. The amino acids in capillary endothelial cells. It inhibits angiogenesis the PEST sequence are positively charged. PEST and tumour growth [4]. In 1993, Tuszynski et al. sequences are thought to be a signal peptide for isolated a new TSP-1-binding protein, angiocidin. protein degradation [1-2]. Another element of It has been shown that a tumour is expressed in protein destruction is the ‘N end rule’ which was excess in the stroma and that a tumour is active discovered in 1986 by Alexander Varshavsky and in the progressive mechanisms. This protein is his colleagues. This rule is related to ubiquitination. described as a potent inhibitor of angiogenesis The time from the N-terminal amino terminal of and tumour growth. It inhibits angiogenesis by a rule protein to its complete destruction is the binding to collagen and collagen receptors. It half-life of this protein. However, the duration of also causes the differentiation of monocytes into the destruction of amino acids in eukaryotic and macrophages in THP1, a cell line of monocytic prokaryotic organisms is changing. Amino acid leukaemia. This differentiation occurs at the same modification and variants found in the N-terminal time with the dramatic changes in cell morphology can cause this difference. However, the half-life and the macrophage marker expression. It has of proteins can be deduced from this rule as an also been reported that angiocidin treatment estimate [3]. may enhance the ability of monocytes to present antigens to T lymphocytes and lead to the The destruction of a protein by the ubiquitin- destruction of tumour cells (directly or indirectly) proteasome pathway begins with two steps: by functional macrophages [11]. the first step is the covalent attachment of the target substrate to multiple ubiquitin chains. Angiocidin protein sequence has a high- The combination of ubiquitin and proteins to be level homology with other proteins (S5a and destroyed may include control, DNA antisecretory factor) (both have identical repair, biosynthesis of ribosome-like organelles, sequences). Angiocidin differs from them with MHC class 1 antigen presentation and inflammatory 3 additional amino acids (located in carboxyl response, endocytosis of cell surface proteins terminus) [5]. There are so many research in the and modulation of ion channels, morphogenesis literature about cancer and this two proteins, S5a of neuronal networks, Nf- Regulation, long-term and Angiocidin. But the working mechanism of memory and biorhythm formation, apoptosis, these proteins could not be explained exactly yet. differentiation, stress response and protein quality. The second step in the ubiquitin- PSMD4 mRNA or protein expression were proteasome pathway is the degradation of the shown in some cell lines, THP-1, i.e. acute ubiquitin-releasing protein, releasing ubiquitin. monocytic leukemia cell line; MCF-7, i.e. breast Some of the ubiquitin system targets receptors of cancer cell line; H1299, i.e. human non-small cell cancer-stimulating growth factors such as N-, lung carcinoma cell line; HeLa, i.e. cervical cancer c-fos, c-jun, Src and EGFR, or even direct tumour cells; Colo320, i.e. Dukes’ type C, colorectal suppressor proteins [10]. adenocarcinoma cell line; LoVo, i.e. Colorectal S. Aydogan Turkoglu et al. / Hacettepe J. Biol. & Chem., 2018, 46 (3), 329–336 335

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